Effects of prolonged ingestion of glucose or ethanol on tissue lipid composition and lipid biosynthesis in rat

The effects on lipid metabolism of long-term feeding of large amounts of ethanol or glucose differed from those that have been reported in short-term experiments. Three groups of male rats were investigated. The first was fed lab chow and 15% (v/v) ethanol ad lib.; the second was pair-fed with the first and given isocaloric amounts of glucose in lieu of ethanol; the third was fed lab chow and water ad lib. All three groups consumed nearly the same number of calories, and about 30% of the calories in the first group were derived from ethanol. Neither glucose nor ethanol added to a nutritionally adequate diet promoted the development of a fatty liver, although both stimulated acetate-(14)C utilization for hepatic lipid synthesis. In all three groups more than 80% of the label in hepatic lipid was found in fatty acids, and the distribution of label amongst the fatty acids of different chain lengths was virtually the same. Ethanol decreased while glucose increased the quantity of lipid in fat depots, and each altered the fatty acid composition of the lipids in adipose tissue, kidney, liver, and hepatic subcellular fractions in a different manner. The most striking of these changes was the relative increase in monounsaturated fatty acids and the decrease in essential fatty acids produced by glucose.     

Dietary linoleic acid is required for development of experimentally induced alcoholic liver injury

We had previously hypothesized that linoleic acid (LA) was essential for development of alcoholic induced liver injury in our rat model. Male Wistar rats were fed a nutritionally adequate diet (25% calories as fat) with ethanol (8-17 g/kg/day). The source of fat was tallow (0.7% LA), lard (2.5% LA) or tallow supplemented with linoleic acid (2.5%). Liver damage was followed monthly by obtaining blood for alanine aminotransferase assay and liver biopsy for assessment of morphologic changes. Enzyme and histologic changes (fatty liver, necrosis and inflammation) in the tallow-linoleic acid-ethanol fed animals were more severe than in the lard-ethanol group. The tallow ethanol group did not show any evidence of liver injury. Our results strongly support our hypothesis that LA is essential for development of alcoholic liver disease in our rat model.     

Both acute and chronic exercise enhance in vivo ethanol clearance in rats

Rates of ethanol clearance were measured at rest and with acute exercise in four groups of female Sprague-Dawley rats. Two groups were trained to run on a motor-driven rodent treadmill at 27 m/min, 1 h/day, 5 days/wk and were given a nutritionally balanced liquid diet; one of these groups received 35% calories as ethanol whereas in the other, sucrose was isocalorically substituted for the ethanol. Appropriate sedentary and nonethanol controls were also used. Clearance of a 1.75-g/kg ethanol dose injected intraperitoneally was determined by measuring ethanol levels in the blood each hour and utilizing these values in the Widmark equation (R. Teschke, F. Moreno, and A. Petrides, Biochem. Pharmacol. 30: 1745-1751, 1981) for calculating whole-body ethanol clearance. Rates of ethanol clearance were determined for each rat at 4 and 7 wk of training. The clearance tests at 4 wk included a 60-min period of running exercise, whereas the tests 3 wk later were conducted at rest. The results indicate that both acute exercise and exercise training can increase rates of in vivo ethanol clearance. In addition, the chronic exercise appeared to increase in vitro ethanol metabolism by hepatic microsomes without altering in vitro hepatic alcohol dehydrogenase activity.     

Effect of chronic intake of ethanol on lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios in rat liver.

Adult male rats were fed a liquid diet providing 35% of the calories as ethanol, while pair-fed controls received the corresponding diet with alcohol replaced by an equicaloric concentration of sucrose. After 1 month, lactate/pyruvate (L/P) and beta-hydroxybutyrate/acetoacetate (beta-HB/AcAc) ratios in the livers were determined under five different conditions: (1) both diets present up to the time of sacrifice, (2) ethanol diet replaced by control diet for 24 h before sacrifice, (3) ethanol diet replaced by control diet for 48 h before sacrifice, (4) as in the preceding, followed by intraperitoneal (i.p.) injection of ethanol, 1 g/kg, 1 h before sacrifice, (5) as in the preceding, but i.p. injection 3 h before sacrifice. The L/P ratio was significantly higher in the alcohol group than in controls under the first experimental condition, but the groups did not differ under the other four conditions. The beta-HB/AcAc ratio was also significantly higher in the alcohol group under the first condition. This difference disappeared in the second and third conditions. Under the fourth and fifth conditions the beta-HB/AcAc ratio was significantly higher in the controls. The results are compatible with an adaptive increase in mitochondrial reoxidation of NADH in the chronic alcohol groups, but the possibility of a change due to alcohol withdrawal can not be excluded.     

Effects of Chronic Ethanol Administration on Rat Brain Phospholipid Metabolism

Alterations in brain phospholipid metabolism were observed after chronic ethanol administration for 16 days to developing rats. Animals were injected intraperitoneally with 32Pi 16 h prior to killing. Overall uptake of 32Pi by brain did not differ between the control and ethanol-treated groups, which were killed 2 h and 24 h after the last ethanol feeding. Except for an increase in the labeling of myelin after ethanol treatment, the amount of radioactivity recovered in the synaptosomal-mitochondrial and plasma membrane fractions of control and ethanol-treated groups was not different. Relative to the radioactivity of phosphatidylcholines, which indicated no change, there were increases (20-44%) in labeling of ethanolamine plasmalogens, phosphatidic acids, and phosphatidylinositols in cortical synaptosomes from the 2-h ethanol-treated group. In the plasma membrane fractions, however, increases (9-14%) in labeling of phosphatidylserines and phosphatidylinositols were observed in both 2- and 24-h ethanol-treated groups. In both membrane fractions, there was an obvious increase (44-86%) in labeling of polyphosphoinositides at 24 h after withdrawal from ethanol. Results thus indicate an adaptive increase in the biosynthesis of ethanolamine plasmalogen and brain acidic phospholipids due to chronic ethanol administration. Furthermore, the increase in labeling of polyphosphoinositides in the 24-h withdrawal group may reflect the hypoactivity associated with ethanol withdrawal.     

Intermittent hypoxia conditioning prevents behavioral deficit and brain oxidative stress in ethanol-withdrawn rats.

Intermittent hypoxia (IH) has been found to protect brain from ischemic injury. We investigated whether IH mitigates brain oxidative stress and behavioral deficits in rats subjected to ethanol intoxication and abrupt ethanol withdrawal (EW). The effects of IH on overt EW behavioral signs, superoxide generation, protein oxidation, and mitochondrial permeability transition pore (PTP) opening were examined. Male rats consumed dextrin or 6.5% (wt/vol) ethanol for 35 days. During the last 20 days, rats were treated with repetitive (5-8 per day), brief (5-10 min) cycles of hypoxia (9.5-10% inspired O2) separated by 4-min normoxia exposures. Cerebellum, cortex, and hippocampus were biopsied on day 35 of the diet or at 24 h of EW. Superoxide and protein carbonyl contents in tissue homogenates and absorbance decline at 540 nm in mitochondrial suspensions served as indicators of oxidative stress, protein oxidation, and PTP opening, respectively. Although IH altered neither ethanol consumption nor blood ethanol concentration, it sharply lowered the severity of EW signs including tremor, tail rigidity, and startle response. Compared with dextrin and ethanol per se, in the three brain regions, EW increased superoxide and protein carbonyl contents and accelerated PTP opening in a manner ameliorated by IH. Administration of antioxidant N-acetylcysteine throughout the IH program abrogated the reductions in EW signs and superoxide content, implicating IH-induced ROS as mediators of the salutary adaptations. We conclude that IH conditioning during chronic ethanol consumption attenuates oxidative damage to the brain and mitigates behavioral abnormalities during subsequent EW. IH-induced ROS may evoke this powerful protection.     

Proliferative Activity of Matrix Cells in the Lateral Ventricles and Granule Cells in the Dentate Gyrus of the Rat Hippocampus under Conditions of Pre- and Postnatal Alcohol Intoxication

The treatment of pregnant and lactating female rats with ethanol inhibits the proliferation of matrix cells in the lateral brain ventricles of fetuses and, during the early postnatal period, granule cells in the dentate gyrus and cells of the ventral horn of Ammon. A low proliferation rate leads to a decrease in the number of neurons forming the granule layer of the dentate gyrus and pyramidal neurons in the CA-1 field of the horn of Ammon.     

Ethanol-ascorbate interrelationship in acute and chronic alcoholism in the guinea pig

The effects of low (200 ppm) and of high (2000 ppm) ascorbic acid, in a nutritionally adequate diet, on blood ethanol levels have been studied in permanently carotid-cannulated, ethanol-infused, unanesthetized guinea pigs. In the acute study, the postinfusion rate of ethanol decline in the blood of animals treated with ascorbic acid was significantly higher when compared with animals treated with fructose, and the rate in the two treated groups was significantly higher than in untreated controls. In the chronic study, animals were infused with sublethal doses of ethanol (30% of the total caloric intake) for 8 weeks. Blood ethanol levels monitored throughout this period showed, at 3 hr postinfusion, a lower concentration in the group on a high ascorbic acid diet. Both experimental groups receiving ethanol lost significantly more body weight in the second week of dieting; but, while the group on high ascorbic acid regained weight steadily thereafter, the group on low ascorbic acid was still 50 g below the controls at the end of the experiment. Liver, kidney, and adrenal ascorbic acid concentrations were lower in the ethanol-treated groups compared to controls. Examination of the liver revealed more fatty metamorphosis or steatosis in the low ascorbic acid group, but there was no evidence of liver fibrosis or cirrhosis. These results demonstrate the feasibility of utilizing the guinea pig for the study of the biochemical and morphological sequelae of alcoholism. They further support the contention that a diet which is nutritionally adequate may no longer be so in the presence of high ethanol intake, and that supplemental vitamin C ingestion may afford protection against ethanol toxicity.     

The effects of chronic alcohol and vitamin E consumption on aging pigments and learning performance in mice

Chronic ethanol consumption further accelerates age-related impairment of shuttle box avoidance learning in mice. The hypothesis was tested that the behavioral impairment is a result of brain lipofuscin pigment deposition, which may be accelerated by ethanol consumption and prevented by the antioxidant effects of pharmacological doses of vitamin E. Feeding an ethanol-containing liquid diet for 5 months did not increase the lipofuscin content when compared with mice pair-fed a liquid diet containing isocaloric amounts of sucrose or standard solid laboratory food containing nutritionally adequate amounts of vitamin E. Supplementation of diets with vitamin E decreased brain lipofuscin content in all groups but failed to prevent the age- or ethanol-induced learning deficit. There was no effect of chronic ethanol consumption on brain weights, DNA, RNA, or protein content.It is concluded that the age-related impairment of avoidance learning is accelerated by chronic alcohol consumption. At the molecular level this acceleration is not caused by an increased brain lipofuscin deposition nor is it prevented by the antioxidant effects of vitamin E.     

Cholestasis following chronic alcohol consumption: Enhancement after an acute dose of chlorpromazine

Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing either 36% of total calories as ethanol or isocaloric carbohydrates (controls) for 4 weeks. Compared to controls, chronic alcohol consumption leads to slightly increased serum activities of various hepatic enzymes including the alkaline phosphatase (AP). Chlorpromazine administered as a single dose 18 hours after ethanol with-drawal resulted within 18 hours in a significant increase of serum AP activity in rats fed ethanol chronically but not in their pair-fed controls. It is concluded that chronic alcohol consumption predisposes to cholestasis due to chlorpromazine.     

Effect of ethanol on androgen receptors in the anterior pituitary, hypothalamus and brain cortex in rats

The purpose of this study was to investigate ethanol-induced changes in androgen receptor sites in the anterior pituitary, hypothalamus, and brain cortex. Young adult male King-Holtzman rats were fed for 5 months a nutritionally complete liquid diet, with ethanol or isocaloric sucrose constituting 36% of the total calories. Androgen receptor sites were measured by sucrose density gradient and charcoal assay using tritiated dihydrotestosterone (DHT). Scatchard plot analysis of the data revealed that apparent dissociation constants of DHT-receptor complex for the anterior pituitary, hypothalamus, and brain cortex from alcohol-fed animals were estimated to be 0.7 +/- 0.13, 0.6 +/- 0.16 and 0.9 +/- 0.15 nM, respectively. These values are identical to those of their isocaloric controls. The concentrations of cytosol androgen receptors of the pituitary, hypothalamus, and brain cortex from alcohol-fed rats were 8.0 +/- 1.2, 6.2 +/- 1.0 and 4.9 +/- 0.7 fmol/mg protein, respectively. This represents about a 34, 24, and 22% reduction when compared to the values of the isocaloric control animals. In contrast to control rats, neither castration nor androgen or LHRH replacement to castrated alcohol-fed rats altered an alcohol-induced reduction of androgen receptor contents. Serum LH and testosterone levels were significantly decreased in alcohol-fed rats but these hormone levels were increased by administration of LHRH or norepinephrine. Such reduction of androgen receptors, serum LH and testosterone, but enhancement of these hormone levels by treatment with neurohormone and neurotransmitter in these animals suggests that ethanol exerts an adverse effect on the hypothalamic-pituitary unit and the neurotransmitter-hypothalamic hormone relationship, resulting in impairment of the androgen-induced sexual events and a suppression of the pituitary gonadotropin secretion.     

Effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption

This study examined the effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption. At the beginning of experiment (week 0), according to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, rats (n = 24) were divided into 3 groups and fed with a standard diet (group C), a diet containing ethanol (group E), or a diet containing ethanol and β‐carotene (group E+B). After 10 weeks, plasma AST and ALT, fat accumulation in the liver, antioxidant enzyme activities in erythrocytes and the liver, malondialdehyde (MDA), and α‐tocopherol and retinol in plasma and hepatic samples were analyzed. The chronic alcohol diet significantly increased AST and ALT levels in plasma, and these changes were prevented by supplementing the diet with β‐carotene. Glutathione (GSH) in erythrocytes and in the liver was significantly elevated in rats fed with a diet containing β‐carotene. The results indicate that β‐carotene supplementation can prevent ethanol‐induced liver damage and increase GSH concentrations in erythrocytes and the liver. Copyright © 2009 John Wiley & Sons, Ltd.     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

Relationship of membrane physical properties to alcohol dependence in mice selected for genetic differences in alcohol withdrawal

Genetic selection based on severity of withdrawal seizures following inhalation of ethanol vapor has produced two lines of mice, WSR (withdrawal seizure resistant) and WSP (withdrawal seizure prone), that differ markedly in withdrawal signs. In the present study, we report that these mice also differed in the severity of withdrawal seizures following consumption of an ethanol-containing liquid diet but did not differ in ethanol intake. In contrast to ethanol withdrawal seizures, the lines displayed similar sensitivity to electrical- or pentylenetetrazole-induced seizures. These results suggest that the lines differ in the development of physical dependence on ethanol rather than seizure sensitivity per se. Because decreased synaptic membrane fluidity has been associated with ethanol dependence, we used fluorescence polarization of diphenylhexatriene and trimethylammonium-diphenylhexatriene to evaluate membrane fluidity in WSP and WSR mice fed lab chow, an ethanol-containing liquid diet, or an isocaloric sucrose-containing liquid diet. Fluidity of brain synaptic membranes was identical for WSP and WSR mice fed lab chow. The control liquid diet did not alter membrane fluidity, and the ethanol diet decreased fluidity equally for WSP and WSR mice. Thus, the genetic difference in development of ethanol dependence found in these lines was not reflected in the physical properties of brain membranes.     

Comparative Effects of Ethanol and Malnutrition on the Development of Catecholamine Neurons: Changes in Neurotransmitter Levels

Abstract: The comparative effects of exposure to ethanol and malnutrition on the concentrations of tyrosine and catecholamines in whole brain and selected regions of brain have been studied in the developing rat. These animals were the offspring of optimally nourished rats (control pups), of rats fed a diet with 35% of the calories supplied by ethanol (ETOH pups), or of animals fed a diet calorically equivalent to the latter but lacking ethanol (iso-caloric, 1C pups). These diets were administered to dams either during the last week of gestation (prenatal) or during lactation (postnatal). Tyrosine levels were elevated prior to birth in the prenatal ETOH or IC pups or at 1 and 2 weeks of age in postnatal ETOH or 1C pups as compared with values found in the control offspring. Dopamine concentration in whole brain was significantly lower in prenatal ETOH pups than in prenatal IC pups at 3 weeks of age. Levels in the brains of postnatal ETOH pups were lower than control values, but not relative to animals exposed to 1C diet. Investigation of corpus striatum showed a significant decrease in dopamine concentration compared with control or IC pup values as a result of postnatal exposure to ethanol. Norepinephrine levels in the whole brain of prenatal ETOH pups were consistently 30–40% lower than either control or matched 1C pups during development. At 3 weeks of age, the norepinephrine levels in the hypothalamus of animals exposed to ethanol pre or postnatally were 30–60% lower than values in the corresponding region in either control or 1C pups. In the rat model described, ethanol caused a decrease in catecholamine levels, perhaps solely by affecting the norepinephrine neurons.     

The effect of the prostaglandin analogue-misoprostol on rat liver mitochondria after chronic alcohol feeding

Rats fed ethanol (36% of total calories in a nutritionally adequate liquid diet) for 5 weeks develop functional alterations of hepatic mitochondria and steatosis of the liver. At the fatty liver stage, ADP-stimulated respiration of mitochondria was depressed in ethanol fed rats by 30% (p less than 0.001) with glutamate + malate and by 23% (p less than 0.001) with succinate as substrates. A similar decrease was noted in the respiratory control ratio (RCR) (34% and 29%, respectively). The total lipid content of the liver increased 2.6 fold (p less than 0.001). Mitochondrial dysfunction could be prevented, in part, by the treatment with a synthetic derivative of prostaglandin E1, misoprostol, at a mean daily dose of 80 micrograms/kg of body weight. The RCR with glutamate + malate as substrates was improved by 36% (p less than 0.05). We conclude that misoprostol attenuates several functional alterations in liver mitochondria during alcohol feeding.     

Omega-3 supplementation can restore glutathione levels and prevent oxidative damage caused by prenatal ethanol exposure

Prenatal ethanol exposure (PNEE) causes long-lasting deficits in brain structure and function. In this study, we have examined the effect of PNEE on antioxidant capacity and oxidative stress in the adult brain with particular focus on four brain regions known to be affected by ethanol: cerebellum, prefrontal cortex and hippocampus (cornu ammonis and dentate gyrus subregions). We have utilized a liquid diet model of fetal alcohol spectrum disorders that is supplied to pregnant Sprague-Dawley rats throughout gestation. To examine the therapeutic potential of omega-3 fatty acid supplementation, a subset of animals were provided with an omega-3-enriched diet from birth until adulthood to examine whether these fatty acids could ameliorate any deficits in antioxidant capacity that occurred due to PNEE. Our results showed that PNEE caused a long-lasting decrease in glutathione levels in all four brain regions analyzed that was accompanied by an increase in lipid peroxidation, a marker of oxidative damage. These results indicate that PNEE induces long-lasting changes in the antioxidant capacity of the brain, and this can lead to a state of oxidative stress. Postnatal omega-3 supplementation was able to increase glutathione levels and reduce lipid peroxidation in PNEE animals, partially reversing the effects of alcohol exposure, particularly in the dentate gyrus and the cerebellum. This is the first study where omega-3 supplementation has been shown to have a beneficial effect in PNEE, reducing oxidative stress and enhancing antioxidant capacity.     

Binge ethanol exposure decreases neurogenesis in adult rat hippocampus

Alcoholism is associated with cognitive deficits and loss of brain mass. Recent studies have indicated that neural progenitor cells proliferate throughout life forming neurons, astrocytes, and oligodendrocytes. The dentate gyrus is one neurogenic region of the adult brain containing neural progenitor cells. To determine if binge ethanol (EtOH) exposure alters neural progenitor cell proliferation and survival, bromodeoxyuridine was administered to adult male rats following an acute or chronic binge exposure paradigm. For an acute binge, rats were gavaged with a 5 g/kg dose of EtOH or vehicle, administered bromodeoxyuridine, and killed either 5 h or 28 days after EtOH treatment. In a 4-day, chronic-binge paradigm, rats were infused with EtOH three times per day (mean dose 9.3 g/kg/day) or isocaloric control diet. Rats were given bromodeoxyuridine once a day for the 4 days of chronic binge treatment, then perfused either immediately following the last dose of EtOH or 28 days later. In both EtOH treatment groups, binge EtOH decreased neural progenitor cell proliferation. Following the chronic four-day binge, neural progenitor cell survival was decreased. These studies are the first to show EtOH inhibition of neural progenitor cell proliferation and survival in the adult, a possible new mechanism underlying alcoholic cognitive dysfunction.     

Effect of chronic carbon monoxide exposure on experimental alcoholic liver injury in rats

Two groups of experimental animals with pair-fed controls were studied to evaluate the effect of chronic carbon monoxide (CO) exposure on progression of experimental alcoholic liver injury. Eight pairs of male Wistar rats were continuously infused liquid diet and ethanol or isocaloric dextrose for four months. Four pairs were also exposed to CO. Liver damage was followed monthly by serum ALT and morphologic assessment of liver biopsy. Serum levels of ALT were significantly higher in the CO-ethanol group compared to other groups. Electron microscopy revealed a greater degree of cell necrosis in the CO exposed group which explained the significantly higher ALT activity in these animals. Both experimental groups (CO-ethanol and air-ethanol) had significantly greater liver damage than controls. Carboxyhemoglobin levels were not different in the ethanol-fed and control group. Our results show that chronic CO exposure enhances liver cell necrosis in ethanol-fed rats thereby lending support to the hypothesis that ethanol and hypoxia enhance cellular disruption in the liver which could be important in the pathogenesis of alcoholic liver disease in rats.     

Toxic effects of combined treatment of 1,2‐dichloroethane and ethanol on mouse brain and the related mechanisms

The aim of this study was to explore the mechanisms of brain damage induced by the combined treatment of mice with 1,2‐dichloroethane (1,2‐DCE) and ethanol. Mice were divided into control group; 1,2‐DCE‐intoxicated group; ethanol‐treated group; and low‐, medium‐, and high‐dose combined treatment groups. Histological observations along with brain organ coefficients and water content were used to measure the brain damage directly and indirectly. The levels of nonprotein sulfhydryls, malondialdehyde (MDA), and superoxide dismutase activity were used as parameters to evaluate oxidative stress in the brain. Protein and messenger RNA (mRNA) levels of cytochrome P450 2E1 (CYP2E1), zonula occludens‐1 (occludin and zo‐1), aquaporin‐4 (AQP4), nuclear factor erythroid 2‐related factor 2 (Nrf2), heme oxygenase (HO)‐1, and the γ‐glutamyl cysteine synthetase catalytic and modulatory subunits (γ‐GCSc, GR, and γ‐GCSm) in the brain were examined by Western blot analysis and quantitative polymerase chain reaction analysis, respectively. Effects of the combined treatment of 1,2‐DCE and ethanol were evaluated by analysis of variance with a factorial design. The results suggested that combined exposure to ethanol and 1,2‐DCE synergistically increased CYP2E1 protein and mRNA levels, accelerated the metabolism of ethanol and 1,2‐DCE in the brain tissue, induced high production of reactive oxygen species (ROS), and increased MDA levels, thereby damaging the blood‐brain barrier and causing obvious pathological changes in brain tissue. However, the increased level of ROS activated the Nrf2 signal transduction pathway, promoting the expression of HO‐1 and glutathione‐related antioxidant enzymes in the brain to protect the cells from oxidative damage.